Polycationic Compounds And Uses Thereof

ABSTRACT

Aspects of the present invention relate to compounds and methods useful in modulating angiogenesis and methods of treating or preventing diseases associated with angiogenesis by administering a polycationic compound. The present invention relates to methods of use and compositions for inhibiting angiogenesis-mediated disorders in mammals including animals and humans. Additionally, this invention relates to the combined use of polycations with other anti-angiogenesis agents for the treatment of different angiogenesis-mediated disorders. Additionally, those polycationic compounds can be used with various anti-inflammatory and cytotoxic agents as well as with radio-therapeutic agents in cancer patients to prevent and treat tumor growth and metastasis.

REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §120 as a continuationof U.S. application Ser. No. 12/720, 734, filed Mar. 20, 2010, which isa continuation of U.S. application Ser. No. 12/465,995, filed May 14,2009, which is a continuation of U.S. application Ser. No. 11/154,962filed Jun. 15, 2005, which claims priority under 35 U.S.C. §119(e) toU.S. Provisional Application Ser. No. 60/579,282 filed on Jun. 15, 2004,each of which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Angiogenesis is the development of new blood vessels from preexistingblood vessels Physiologically, angiogenesis ensures proper developmentof mature organisms, prepares the womb for egg implantation, and plays akey role in wound healing, fracture repair, and the establishment andmaintenance of pregnancy. Angiogenesis is also associated withpathological conditions associated with a number of disease states suchas cancer, inflammation, and ocular diseases.

Angiogenesis or “neovascularization” is a multi-step process controlledby the balance of pro- and anti-angiogenic factors. The latter stages ofthis process involve proliferation and organization of endothelial cells(EC) into tube-like structures. Growth factors such as fibroblast growthfactor 2 (FGF2) and vascular endothelial growth factor (VEGF) arethought to be key players in promoting endothelial cell growth anddifferentiation. The endothelial cell is the pivotal component of theangiogenic process and responds to many cytokines through its cellsurface receptors and intracellular signaling mechanisms.

Control of angiogenesis is a complex process involving local release ofvascular growth factors, extracellular matrix adhesion molecules, andmetabolic factors. Mechanical forces within blood vessels may also playa role. The principal classes of endogenous growth factors implicated innew blood vessel growth are the fibroblast growth factor (FGF) familyand vascular endothelial growth factor (VEGF). The mitogen-activatedprotein kinase (MAPK; ERK 1/2) signal transduction cascade is involvedboth in VEGF gene expression and in control of proliferation of vascularendothelial cells.

Many diseases and undesirable conditions could be prevented oralleviated if it were possible to stop the growth and extension ofcapillary blood vessels under some conditions, at certain times, or inparticular tissues. Angiogenesis-dependent diseases that can be treatedby the invention disclosed herein are those conditions/diseases whichrequire or induce vascular growth. On the other hand, promotion ofangiogenesis is desirable in situations where vascularization is to beestablished or extended, such as, but not limited to, stroke, heartdisease, ulcers, scleroderma and infertility.

It has been proposed that inhibition of angiogenesis would be a usefultherapy for restricting the unregulated growth of blood vessels, forexample, in tumor growth. Inhibition of angiogenesis can be achieved byinhibiting endothelial cell response to angiogenic stimuli as suggestedby Folkman et al., Cancer Biology 3:89-96 (1992), where examples ofendothelial cell response inhibitors such as angiostatic steroids,fungally derived products such as fumagilin, platelet factor 4,thrombospondin, alpha-interferon, vitamin D analogs, and D-penicillamineare described. For additional proposed inhibitors of angiogenesis, seeBlood et al., Bioch. Biophys. Acta 1032:89-118 (1990), Moses et al.,Science 248:1408-1410 (1990), and U.S. Pat. Nos. 5,092,885, 5,112,946,5,192,744, and 5,202,352.

Inhibiting an undesired angiogenic processes may provide a therapeutictreatment and/or preventive against inappropriate or undesiredangiogenesis. Conversely, promoting an angiogenic process may provide atherapeutic treatment for those diseases states that would benefit fromangiogenesis. Aspects of the invention disclosed herein provideamphiphilic compounds, such as polycationic compounds, for theiranti-angiogenic properties. The ability to inhibit angiogenesis mayprovide an effective therapeutic tool for modulating angiogenic diseasesand/or conditions.

SUMMARY OF THE INVENTION

An aspect of the present invention relates to polycationic compounds andcompositions containing polycationic compounds useful in modulatingangiogenesis.

Polycationic

Another aspect of the present invention relates to a method ofmodulating angiogenesis in an animal or human in need thereof comprisingadministering to said animal a therapeutically effective amount of apolycationic compound.

A further aspect of the present invention also relates to a method oftreating or preventing a disease or disorder in an animal or human inneed thereof, comprising administering to said animal a therapeuticallyeffective amount of an polycationic compound.

DESCRIPTION OF FIGURES

The file of this patent contains at least one photograph or drawingexecuted in color. Copies of this patent with color drawing(s) orphotograph(s) will be provided by the Patent and Trademark Office uponrequest and payment of necessary fee.

FIG. 1 depicts the effect of polycationic compounds in the CAM model ofangiogenesis.

FIG. 2 is a bar graph depicting the percent inhibition of Factor Xa ofpolycationic compounds of the present invention.

FIG. 3 is a graph depicting the effect of Compound 26, a polycationiccompound of the present invention, on coagulation time.

DESCRIPTION OF THE INVENTION

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to the particularmolecules, compositions, methodologies or protocols described, as thesemay vary. It is also to be understood that the terminology used in thedescription is for the purpose of describing the particular versions orembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must also be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference toa “cell” is a reference to one or more cells and equivalents thereofknown to those skilled in the art, and so forth. Unless definedotherwise, all technical and scientific terms used herein have the samemeanings as commonly understood by one of ordinary skill in the art.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of embodimentsof the present invention, the preferred methods, devices, and materialsare now described. All publications mentioned herein are incorporated byreference. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

As used herein, the term “about” means plus or minus 10% of thenumerical value of the number with which it is being used. Therefore,about 50% means in the range of 45%-55%.

The terms “angiogenesis” or “neovascularization” refer to the generationof new blood supply, e.g., blood capillaries, vessels, and veins, fromexisting blood vessel tissue (e.g., vasculature). The process ofangiogenesis can involve a number of tissue cell types including, forexample, endothelial cells which form a single cell layer lining of allblood vessels and are involved with regulating exchanges between thebloodstream and the surrounding tissues. New blood vessels(angiogenesis) can develop from the walls of existing small vessels bythe outgrowth of endothelial cells. Angiogenesis is also involved intumor growth as it provides tumors with blood supply necessary for tumorcell survival and proliferation (growth).

The terms “patient” and “subject” mean all animals including humans.Examples of patients or subjects include humans, cows, dogs, cats,goats, sheep, and pigs.

A “therapeutically effective amount” in reference to pharmaceuticalcompositions is an amount sufficient to decrease or prevent the symptomsassociated with a medical condition or infirmity, to normalize bodyfunctions in disease or disorders that result in impairment of specificbodily functions, or to provide improvement in one or more of theclinically measured parameters of the disease. As related to the presentapplication, a therapeutically effective amount of a polycationiccompound is an amount sufficient to decrease or inhibit angiogenesis.

Aspects of the present invention relate to methods of modulatingangiogenesis in an animal in need thereof comprising administering tothe animal a therapeutically effective amount of a polycationiccompound. Aspects of the present invention also relate to methods oftreating or preventing a disease or disorder in an animal in needthereof, comprising administering to the animal a therapeuticallyeffective amount of a polycationic compound.

Generally, the polycationic compounds preferably exhibit a rigid orsemi-rigid backbone, such that the structure is torsionally-constrained,that displays positively-charged sidegroups on one face of the backbone.The positively charged sidegroups are optimally distributed along thelength of the backbone and optimally separated from the backbone by acarbon spacer that may optionally contain one or more heteroatoms.Torsional freedom along the backbone may be stabilized by intramolecularhydrogen bonding, steric constraints or cyclization. While preferredformulas of various polycationic compounds, such as arylamides,hydrazides, calixrenes and salicylamides, are described, other suitablesidegroups that may be used to stabilize the positive charge include,for example, (A-G):

Other amines that may be used include, for example, piperidine,4-aminopyridine, morpholine, and aminothiazole. Optionally, the basicityof an amino group may be modulated by incorporating 1 or 2 fluorines onone of the methylene groups in the chain. Other center ring substituentsthat may also be used to rigidify (reduce torsional freedom) of thebackbone include, for example, (H-K):

In one embodiment of the invention, the polycationic compound is anarylamide oligomer compound of the formula:

wherein

X is O or S;

R₁ is C₁-C₉ straight or branched chain alkyl,wherein R₁ is optionally substituted with one or more —NH₂ or

Y is a bond or

Z is a bond or

R₂ is hydrogen or C₁-C₉ straight or branched chain alkyl;wherein said R₂ is optionally substituted with one or more —NH₂ or

or R₂ is —X—R₁; R₃ is

or methylene,wherein said methylene is substituted with C₁-C₉ straight or branchedchain alkyl, wherein said C₁-C₉ straight or branched chain alkyl isoptionally substituted with one or more —NH₂ or

n is 2-10; andm is 1 or 2.

In another embodiment of the invention, the arylamide oligomer compoundis a compound of the formula:

wherein R₁ is

In a preferred embodiment, the compound is an arylamide of the formula:

X is O or S; Y is O or S;

R₁ is H or —C(═O)-A, A C₁ to C₉ straight or branched alkyl, where A isoptionally substituted with one or more —NH₂, —N(CH₃)₂ or

R₂ is C₁ to C₉ straight or branched alkyl, where R₂ is optionallysubstituted with one or more —NH₂, —N(CH₃)₂ or

R₃ is C₁ to C₉ straight or branched alkyl, where R₃ is optionallysubstituted with one or more —NH₂, —N(CH₃)₂ or

R₄ is H, -B or —C(═O)—O-B, where B is C₁ to C₉ straight or branchedalkyl.

In another embodiment, the polycationic compound is a hydrazide of theformula:

whereinn=1 to 10;

X is O or S; Y is O or S;

Z is a bond, C₁ to C₉ straight or branched alkyl, or a 1,4-cyclohexylR₁ is NH₂ or NH-A, where A is C₁ to C₉ straight or branched alkyl, whereA is optionally substituted with —NH₂, —N(CH₃)₂ or

R₂ is C₁ to C₉ straight or branched alkyl, where R₂ is optionallysubstituted with one or more —NH₂, —N(CH₃)₂ or

R₃ is C₁ to C₉ straight or branched alkyl, where R₃ is optionallysubstituted with one or more —NH₂, —N(CH₃)₂ or

R₄ is H or

In another embodiment of the invention, the polycationic compound is acalixrene of the formula:

whereinn=2-8, more preferably 4-8;X is a bond, O or —O—CH₂—C(═O)—O—,R₁ is −A or —O-A, where A is C₁ to C₉ straight or branched alkyl;R₂ is C₁ to C₉ straight or branched alkyl, where R₂ is optionallysubstituted with one or more —NH₂, —N(CH₃)₂ or

In another embodiment of the invention, the polycationic compound is asalicylamide of the formula:

n is 2 to 10;

R₁ is H or

R₂ is C₁ to C₉ straight or branched alkyl, where R₂ is optionallysubstituted with one or more —NH₂, —N(CH₃)₂ or

R₃ is C₁ to C₉ straight or branched alkyl, where R₂ is optionallysubstituted with one or more —NH₂, —N(CH₃)₂ or

R₄ is OH, NH₂ or

where A is OH or NH₂.

In a further embodiment, the polycationic compound is selected from thegroup consisting of Compound 1, Compound 2, Compound 3, Compound 4,Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10,Compound 11, Compound 12, Compound 13, Compound 14, Compound 15,Compound 16, Compound 17, Compound 18, Compound 19, Compound 20,Compound 21, Compound 22, Compound 23, Compound 24, Compound 25,Compound 26, Compound 27, Compound 28, Compound 29, Compound 30,Compound 31, Compound 32, Compound 33, Compound 34, Compound 35,Compound 26, Compound 37, Compound 38, Compound 39, Compound 40,Compound 41, Compound 42, Compound 43, Compound 44, Compound 45,Compound 46, Compound 47, Compound 48, Compound 49, Compound 50,Compound 51, Compound 52, Compound 53, Compound 54 and Compound 55, asdepicted below in Table 1.

While Compound 1 and Compound 2 comprise the same arylamide structure,Compound 1 and 2 are synthesized under different conditions and exhibitdifferent purities. During polymerization, DCM (dichloromethane) wasused as a solvent for Compound 2 and NMP (1-methyl-2-pyrrolidinone) wasused for Compound 1. Polymerization is less efficient in NMP, thereforeCompound 1 is n=2-10 and n=10 for Compound 2.

TABLE 1 Polycationic Compounds. Compound Structure Compound 1

Compound 2

Compound 3

Compound 4

Compound 5

Compound 6

Compound 7

Compound 8

Compound 9

Compound 10

Compound 11

Compound 12

Compound 13

Compound 14

Compound 15

Compound 16

Compound 17

Compound 18

Compound 19

Compound 20

Compound 21

Compound 22

Compound 23

Compound 24

Compound 25

Compound 26

Compound 27

Compound 28

Compound 29

Compound 30

Compound 31

Compound 32

Compound 33

Compound 34

Compound 35

Compound 36

Compound 37

Compound 38

Compound 39

Compound 40

Compound 41

Compound 42

Compound 43

Compound 44

Compound 45

Compound 46

Compound 47

Compound 48

Compound 49

Compound 50

Compound 51

Compound 52

Compound 53

Compound 54

Compound 55

In more preferred embodiments, the polycationic compound comprisesCompound 26, Compound 28, Compound 29, Compound 34, Compound 48, orCompound 50 or a combination thereof.

Other polycationic compounds useful in the methods of the presentinvention are those described in WO 02/072007 entitled “FaciallyAmphiphilic Polymers as Anti-Infective Agents” filed Mar. 7, 2002, WO02/100295 entitled “Facially Amphiphilic Polymers as Anti-InfectiveAgents” filed Mar. 7, 2002 and WO 04//082634 entitled “FaciallyAmphiphillic Polymers and Oligomers and Uses Thereof” filed on Mar. 17,2004.

In another embodiment, a compound is provided for modulatingangiogenesis comprising a therapeutically effective amount of apolycationic compound.

In one embodiment of the invention, the polycationic compound is anarylamide oligomer compound of the formula:

wherein

X is O or S;

R₁ is C₁-C₉ straight or branched chain alkyl,wherein R₁ is optionally substituted with one or more —NH₂ or

Y is a bond or

Z is a bond or

R₂ is hydrogen or C₁-C₉ straight or branched chain alkyl;wherein said R₂ is optionally substituted with one or more —NH₂ or

or R₂ is —X—R₁; R₃ is

or methylene,wherein said methylene is substituted with C₁ to C₉ straight or branchedchain alkyl, wherein said C₁ to C₉ straight or branched chain alkyl isoptionally substituted with one or more —NH₂ or

n is 2-10; andm is 1 or 2.

In a preferred embodiment, the compound is an arylamide of the formula:

X is O or S; Y is O or S;

R₁ is H or —C(═O)-A, A is C₁ to C₉ straight or branched alkyl, where Ais optionally substituted with one or more —NH₂, —N(CH₃)₂ or

R₂ is C₁ to C₉ straight or branched alkyl, where R₂ is optionallysubstituted with one or more —NH₂, —N(CH₃)₂ or

R₃ is C₁ to C₉ straight or branched alkyl, where R₃ is optionallysubstituted with one or more —NH₂, —N(CH₃)₂ or

R₄ is H, -B or —C(═O)—O-B, where B is C₁ to C₉ straight or branchedalkyl.

In another embodiment, the polycationic compound is a hydrazide of theformula:

n=1 to 10;

X is O or S; Y is O or S;

Z is a bond, C₁ to C₉ straight or branched alkyl, or a 1,4-cyclohexylR₁ is NH₂ or NH-A, where A is C₁ to C₉ straight or branched alkyl, whereA is optionally substituted with —NH₂, —N(CH₃)₂ or

R₂ is C₁ to C₉ straight or branched alkyl, where R₂ is optionallysubstituted with one or more —NH₂, —N(CH₃)₂ or

R₃ is C₁ to C₉ straight or branched alkyl, where R₃ is optionallysubstituted with one or more —NH₂, —N(CH₃)₂ or

R₄ is H or

In another embodiment, the polycationic compound is a calixrene of theformula:

n=2-8, more preferably 4-8;X is a bond, O or —O—CH₂—C(═O)—O—,R₁ is -A or —O-A, where A is C₁ to C₉ straight or branched alkyl;R₂ is C₁ to C₉ straight or branched alkyl, where R₂ is optionallysubstituted with one or more —NH₂, —N(CH₃)₂ or

In another embodiment of the invention, the polycationic compound is asalicylamide of the formula:

n is 2 to 10;

R₁ is H or

R₂ is C₁ to C₉ straight or branched alkyl, where R₂ is optionallysubstituted with one or more —NH₂, —N(CH₃)₂ or

R₂ is C₁ to C₉ straight or branched alkyl, where R₂ is optionallysubstituted with one or more —NH₂, —N(CH₃)₂ or

R₄ is OH, NH₂ or

where A is OH or NH₂.

In preferred embodiments, the compound comprises Compound 1, Compound 2,Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8,Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound14, Compound 15, Compound 16, Compound 17, Compound 18, Compound 19,Compound 20, Compound 21, Compound 22, Compound 23, Compound 24,Compound 25, Compound 26, Compound 27, Compound 28, Compound 29,Compound 30, Compound 31, Compound 32, Compound 33, Compound 34,Compound 35, Compound 26, Compound 37, Compound 38, Compound 39,Compound 40, Compound 41, Compound 42, Compound 43, Compound 44,Compound 45, Compound 46, Compound 47, Compound 48, Compound 49,Compound 50, Compound 51, Compound 52, Compound 53, Compound 54,Compound 55, or a combination thereof.

In more preferred embodiments, the compound comprises Compound 26,Compound 28, Compound 29, Compound 34, Compound 48, or Compound 50 or acombination thereof.

Salicylamide polycationic compounds may be synthesized as follows:

In another embodiment, a compound is provided for modulatingangiogenesis comprising a therapeutically effective amount of apolycationic compound.

In one embodiment, the compound may contain a therapeutically effectiveamount of a polycationic compound to promote angiogenesis. In apreferred embodiment, the compound may contain a therapeuticallyeffective amount of a polycationic compound to inhibit angiogenesis.

In a preferred embodiment, the compound is an arylamide of the formula:

X is O or S; Y is O or S;

R₁ is H or —C(═O)-A, A is C₁ to C₉ straight or branched alkyl, where Ais optionally substituted with one or more —NH₂, —N(CH₃)₂ or

R₂ is C₁ to C₉ straight or branched alkyl, where R₂ is optionallysubstituted with one or more —NH₂, —N(CH₃)₂ or

R₃ is C₁ to C₉ straight or branched alkyl, where R₃ is optionallysubstituted with one or more —NH₂, —N(CH₃)₂ or

R₄ is H, -B or —C(↑O)—O-B, where B is C₁ to C₉ straight or branchedalkyl.

In another embodiment, the polycationic compound is a hydrazide of theformula:

n=1 to 10;

X is O or S; Y is O or S;

Z is a bond, C₁ to C₉ straight or branched alkyl, or a 1,4-cyclohexylR₁ is NH₂, or NH-A, where A is C₁ to C₉ straight or branched alkyl,where A is optionally substituted with —NH₂, —N(CH₃)₂ or

R₂ is C₁ to C₉ straight or branched alkyl, where R₂ is optionallysubstituted with one or more —NH₂, —N(CH₃)₂ or

R₃ is C₁ to C₉ straight or branched alkyl, where R₃ is optionallysubstituted with one or more —NH₂, —N(CH₃)₂ or

R₄ is H or

In another embodiment of the invention, the polycationic compound is acalixarene of the formula:

n=2-8, more preferably 4-8;X is a bond, O or —O—CH₂—C(═O)—O—,R₂ is -A or —O-A, where A is C₁ to C₉ straight or branched alkyl;R₂ is C₁ to C₉ straight or branched alkyl, where R₂ is optionallysubstituted with one or more —NH₂, —N(CH₃)₂ or

In another embodiment of the invention, the polycationic compound is asalicylamide of the formula:

n is 2 to 10;

R₁ is H or

R₂ is C₁ to C₉ straight or branched alkyl, where R₂ is optionallysubstituted with one or more —NH₂, —N(CH₃)₂ or

R₃ is C₁ to C₉ straight or branched alkyl, where R₂ is optionallysubstituted with one or more —NH₂, —N(CH₃)₂ or

R₄ is OH, NH₂, or

where A is OH or NH₂.

In one aspect of the present invention, the polycationic compounds ofthe present invention may be useful in the treatment of a disease ordisorder associated with angiogenesis. In preferred embodiments, thecompound comprises a therapeutically effective amount of Compound 26,Compound 28, Compound 29, Compound 34, Compound 48, or Compound 50 or acombination thereof.

In another embodiment of the invention, the polycationic compounds maybe used to treat or prevent a disease or disorder associated withinsufficient angiogenesis. Such diseases include, for example, stroke,heart disease, ulcers, infertility and scleroderma.

In one embodiment of the invention, the polycationic compounds may beused to treat or prevent a disease or disorder associated with excessiveangiogenesis. Such diseases include, for example, cancer, rheumatoidarthritis, AIDS complications, psoriasis and blindness.

Generally, cancer refers to any malignant growth or tumor caused byabnormal and uncontrolled cell division; it may spread to other parts ofthe body through the lymphatic system or the blood stream. Cancerinclude both solid tumors and blood-borne tumors. Solid tumors include,or example, but not limited to Kaposi's sarcoma, hemangiomas, solidtumors, blood-borne tumors, breast cancer, lung cancer, ovarian cancer,testicular cancer, colon cancer, rhabdomyosarcoma, retinoblastoma,Ewing's sarcoma, neuroblastoma, and osteosarcoma. Angiogenesis is alsoassociated with blood-borne tumors, such as leukemias, any of variousacute or chronic neoplastic diseases of the bone marrow in whichunrestrained proliferation of white blood cells occurs, usuallyaccompanied by anemia, impaired blood clotting, and enlargement of thelymph nodes, liver and spleen. It is believed to that angiogenesis playsa role in the abnormalities in the bone marrow that give rise toleukemia-like tumors.

In yet another embodiment of the invention, the disease or disorder islung cancer, breast cancer, prostate cancer, colon cancer, renal cancer,bladder cancer, pancreatic cancer, glioblastoma, neuroblastoma,blindness, macular degeneration, diabetic retinopathy, cornealtransplant, myopic degeneration, complications related to AIDS,arthritis, rheumatoid arthritis, psoriasis, scleroderma, inflammatorybowel disease, stroke, heart disease, ulcers and infertility. Forexample, but not limited to, cancers, inflammatory arthritis (such asrheumatoid arthritis), diabetic retinopathy, as well as otherneovascular diseases of the eye (or example, corneal neovascularization,neovascular glaucoma, retrolental fibroblasia and macular degeneration),arteriovenous malformations, conditions of excessive bleeding(menorrhagia), Osler-Webber Syndrome, myocardial angiogenesis, plaqueneovascularization, telangiectasia, hemophiliac joints, angiofibroma,and wound granulation. The anti-angiogenic compositions provided hereinare also useful in the treatment of diseases of excessive or abnormalstimulation of endothelial cells. These diseases include, but are notlimited to, intestinal adhesions, Crohn's disease, atherosclerosis,scleroderma, and hypertrophic scars (i.e., keloids).

Other suitable angiogenesis-mediated disorders that may be treated orprevented with the polycationic compounds provided include, but are notlimited to, tumors and cancer associated disorders (e.g., retinal tumorgrowth, benign tumors (e.g., hemangiomas, acoustic neuromas,neurofibromas, trachomas, and pyogenic granulomas), solid tumors, bloodborne tumors (e.g., leukemias, angiofibromas, and kaposi sarcoma), tumormetastases, and other cancers which require neovascularization tosupport tumor growth), ocular neovascular-disorders (e.g., diabeticretinopathy, macular degeneration, retinopathy of prematurity,neovascular glaucoma, corneal graft rejection, and other ocularangiogenesis-mediated disorders), inflammatory disorders (e.g., immuneand non-immune inflammation, rheumatoid arthritis, chronic articularrheumatism, inflammatory bowel diseases, psoriasis, and other chronicinflammatory disorders), endometriosis, other disorders associated withinappropriate or inopportune invasion of vessels (e.g., retrolentalfibroplasia, rubeosis, and capillary proliferation in atheroscleroticplaques and osteoporosis), Osler-Webber Syndrome, myocardialangiogenesis, plaque neovascularization, telangiectasia, hemophiliacjoints, and wound granulation. Other diseases in which angiogenesisplays a role in the maintenance or progression of the pathological stateare known to those skilled in the art and are similarly intended to beincluded within the meaning of the term angiogenesis-mediated usedherein.

In one embodiment, the polycationic compounds are used in conjunctionwith other angiogenesis inhibitors. Angiogenic inhibitors are known inthe art and can be prepared by known methods. For a description ofangiogenic inhibitors and targets sec, for example, Chen et al., CancerRes. 55:4230-4233 (1995), Good et al., Proc. Natl. Acad. Sci. USA87:6629-6628 (1990), O'Reilly et al., Cell 79:315-328 (1994), Parangi etal., Proc. Natl. Acad. Sci. USA 93:2002-2007 (1996), Rastinejad et al.,Cell 56:345-355 (1989), Gupta et al., Proc. Natl. Acad. Sci. USA92:7799-7803 (1995), Maione et al., Science 247:77-79 (1990), Angiolilloet al., J. Exp. Med. 182:155-162 (1995), Strieter et al., Biochem.Biophys. Res. Comm. 210:51-57 (1995); Voest et al., J. Natl. CancerInst. 87:581-586 (1995), Cao et al., J. Exp. Med. 182:2069-2077 (1995),and Clapp et al., Endocrinology 133:1292-1299 (1993), which are herebyincorporated by reference in their entirety. For a description ofadditional angiogenic inhibitors see, for example. Blood et al., Bioch.Biophys Acta., 1032:89-118 (1990), Moses et al., Science, 248:1408-1410(1990), Ingber et al., Lat Invest., 59:44-51 (1988), and U.S. Pat. Nos.5,092,885 and 5,112,946, which are hereby incorporated by reference intheir entirety.

In another embodiment, the polycationic compounds are used inconjunction with other therapies, such as standard anti-inflammatorytherapies, standard ocular therapies, standard dermal therapies,radiotherapy, tumor surgery, and conventional chemotherapy directedagainst solid tumors and for the control of establishment of metastases.The administration of the angiogenesis inhibitor is typically conductedduring or after chemotherapy at time where the tumor tissue shouldrespond to toxic assault by inducing angiogenesis to recover by theprovision of a blood supply and nutrients to the tumor tissue.Additionally, it is preferred to administer such angiogenesis inhibitorsafter surgery where solid tumors have been removed as a prophylaxisagainst metastasis. Cytotoxic or chemotherapeutic agents are those knownin the art such as aziridine thiotepa, alkyl sulfonate, nitrosoureas,platinum complexes, NO classic alkylators, folate analogs, purineanalogs, adenosine analogs, pyrimidine analogs, substituted urea,antitumor antibiotics, microtubulle agents, and asprignase.

Another aspect of this invention relates to the use of polycationiccompounds in the inhibition of angiogenesis-mediated processes alone orin combination with other existing anti-inflammatory, anti-angiogenesis,anti-cancer, and ocular therapies. Polycationic compounds represent aneffective strategy for the prevention and treatment ofangiogenesis-mediated disorders in cancer, inflammatory, and oculardiseases.

The compounds described above may be administered in a formulationincluding polycationic compounds and derivatives together with anacceptable carrier for the mode of administration. Any formulation ordrug delivery system containing the active ingredients, which issuitable for the intended use, as are generally known to those of skillin the art, can be used. Suitable pharmaceutically acceptable carriersfor oral, rectal, topical or parenteral (including subcutaneous,intraperitoneal, intramuscular and intravenous) administration are knownto those of skill in the art. The carrier must be pharmaceuticallyacceptable in the sense of being compatible with the other ingredientsof the formulation and not deleterious to the recipient thereof.

Formulations suitable for parenteral administration conveniently includesterile aqueous preparation of the active compound, which is preferablyisotonic with the blood of the recipient. Thus, such formulations mayconveniently contain distilled water, 5% dextrose in distilled water orsaline. Useful formulations also include concentrated solutions orsolids containing the compound of formula (I), which upon dilution withan appropriate solvent give a solution suitable for parentaladministration above.

For enteral administration, a compound can be incorporated into an inertcarrier in discrete units such as capsules, cachets, tablets orlozenges, each containing a predetermined amount of the active compound;as a powder or granules; or a suspension or solution in an aqueousliquid or non-aqueous liquid, e.g., a syrup, an elixir, an emulsion or adraught. Suitable carriers may be starches or sugars and includelubricants, flavorings, binders, and other materials of the same nature.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active compound in a free-flowingform, e.g., a powder or granules, optionally mixed with accessoryingredients, e.g., binders, lubricants, inert diluents, surface activeor dispersing agents. Molded tablets may be made by molding in asuitable machine, a mixture of the powdered active compound with anysuitable carrier.

A syrup or suspension may be made by adding the active compound to aconcentrated, aqueous solution of a sugar, e.g., sucrose, to which mayalso be added any accessory ingredients. Such accessory ingredients mayinclude flavoring, an agent to retard crystallization of the sugar or anagent to increase the solubility of any other ingredient, e.g., as apolyhydric alcohol, for example, glycerol or sorbitol.

Formulations for rectal administration may be presented as a suppositorywith a conventional carrier, e.g., cocoa butter or Witepsol S55(trademark of Dynamite Nobel Chemical, Germany), for a suppository base.

Alternatively, the compound may be administered in liposomes ormicrospheres (or microparticles). Methods for preparing liposomes andmicrospheres for administration to a patient are well known to those ofskill in the art. U.S. Pat. No. 4,789,734, the contents of which arehereby incorporated by reference, describes methods for encapsulatingbiological materials in liposomes. Essentially, the material isdissolved in an aqueous solution, the appropriate phospholipids andlipids added, along with surfactants if required, and the materialdialyzed or sonicated, as necessary. A review of known methods isprovided by G. Gregoriadis, Chapter 14, “Liposomes,” Drug Carriers inBiology and Medicine, pp. 287-341 (Academic Press, 1979).

Micro-spheres or nano-spheres formed of polymers or proteins are wellknown to those skilled in the art, and can be tailored for passagethrough the gastrointestinal tract directly into the blood stream.Alternatively, the compound can be incorporated and themicro-spheres/nano-spheres, or composite of both, implanted for slowrelease over a period of time ranging from days to months. See, forexample, U.S. Pat. Nos. 4,906,474, 4,925,673 and 3,625,214, and Jein,TIPS19:155-157 (1998), the contents of which are hereby incorporated byreference.

The polycationic compounds of the present invention exhibitanti-angiogenic effects in vitro and in vivo. In addition, thepolycationic compounds of the present invention exhibit antagonisticeffects against heparin. While not wishing to be bound by theory, theanti-angiogenic effects of the polycationic compounds may be due, atleast in part to the polycationic compounds ability to antagonizeheparin's role in facilitating activation of FGF and VEGF receptors.

For the following examples, various materials were obtained as follows.All reagents were chemical grade and purchased from Sigma Chemical Co.(St. Louis, Mo.) or through VWR Scientific (Bridgeport, N.J.). Cortisoneacetate, bovine serum albumin (BSA), and gelatin solution (2% type Bfrom bovine skin) were purchased from Sigma Chemical Co. (St. Louis,Mo.). M199 growth medium with Earl's salts, basic FGF,Insulin-Transferrin-Selenium-G Supplement (1-T-Se) 100×, Dulbecco'sphosphate buffered salt solution (PBS) with and without Ca⁺² and Mg⁺²,and 0.5 M EDTA were obtained from Gibco BRL (Grand Island, N.Y.). Humanumbilical vein endothelial cells (HUVEC), endothelial cell basal medium(serum-free, EBM), endothelial growth medium (EGM) (supplemented withgrowth factors, fetal calf serum), and 0.025% trypsin/0.01% EDTAsolution were purchased from Clonetics Inc. (San Diego, Calif.). Humanprostrate (TSU-Pr) tumor cells were obtained from American Type CultureCollection (Rockville, Md.). Matrigel® matrix and human collagen typeIII were purchased from Becton Dickinson (Bedford, Mass.). HEMA-3fixative and staining solutions were purchased from BiochemicalSciences, Inc. (Swedesboro, N.J.). Fertilized chicken eggs werepurchased from Charles River Laboratories, SPAFAS Avian Products &Services (North Franklin, Conn.). In vivo neovascularization wasexamined by the method previously described by Auerbach et al. (Auerbachet al., J. Dev. Biol., 41:391-394 (1974), which is hereby incorporatedby reference in its entirety).

Example 1

The following example illustrates the anti-angiogenic effect ofexemplary polycationic compounds of the present invention. Ten-day oldembryos were purchased from Spafas, Inc. (Preston, Conn.) and wereincubated at 37° C. with 55% relative humidity. In the dark with thehelp of a candling lamp, a small hole was punctured in the shellconcealing the air sac with a hypodermic needle. A second hole waspunctured in the shell on the broadside of the egg directly over anavascular portion of the embryonic membrane, as observed duringcandling. A false air sac was created beneath the second hole by theapplication of negative pressure to the first hole, which caused thechorioallantoic membrane (CAM) to separate from the shell. A window,approximately 1.0 cm², was cut in the shell over the dropped CAM withthe use of a small crafts grinding wheel (Dremel, Division of EmersonElectric Company Racine, Wis.) which allowed direct access to theunderlying CAM. Filter disks of #1 filter paper (Whatman International,United Kingdom) were soaked in 3 mg/mL cortisone acetate (Sigma, St.Louis, Mo.) in a solution of 95% ethanol and water and subsequently airdried under sterile conditions. FGF2 (Life Technologies, Gaithersburg,Md.) was used to grow vessels on the CAMs of 10 day old chick embryos.Sterile filter disks adsorbed with FGF2 dissolved in PBS at 1 μg/mL wereplaced on growing CAMs. Sterile filter disks adsorbed with FGF2 wasdissolved in PBS at 1 μg/mL were placed on growing CAM. At 24 h, testcompounds or control vehicle was added directly to CAM topically.

CAM tissue directly beneath FGF2-saturated filter disk was resected fromembryos treated 48 hours prior with test compound or control. Tissueswere washed three times with PBS. Sections were placed in a 35-mm petridish (Nalgen Nunc, Rochester, N.Y.) and examined under a SV6stereomicroscope (Karl Zeiss, Thornwood, N.Y.) at 50× magnification.Digital images of CAM sections adjacent to filters were collected usinga 3-CCD color video camera system (Toshiba America, New York, N.Y.) andanalyzed using Image-Pro Plus software (Media Cybernetics, SilverSpring, Md.). Table 4 contains the number of vessel branch pointscontained in a circular region equal to the area of a filter diskcounted for each section.

CAM tissue directly beneath FGF2-saturated filter disk was resected fromembryos treated 48 h prior with compound or control. Tissues were washedthree times with PBS. Sections were placed in a 35-mm petri dish (NalgeNunc, Rochester, N.Y.) and examined under a SV6 stereomicroscope (KarlZeiss, Thornwood, N.Y.) at 50× magnification. Digital images of CAMsections adjacent to filters were collected using a 3-CCD color videocamera system (Toshiba America, New York, N.Y.) and analyzed with theImage-Pro Plus software (Media Cybernetics, Silver Spring, Md.). Theeffects of polycationic compounds on angiogenesis are shown in Tables 2,3 and 4. The effects are also shown in FIG. 1.

TABLE 2 Anti-angiogenesis efficacy of polycationic compounds in CAMmodel Branch pts ± % Inhibition ± CAM Treatment SEM SEM PBS (control)  69 ± 16.0 FgF (1.0 ug/ml) 155 ± 10 Compound 29 82 ± 5 85 ± 6 (1.2ug) + FGF2 (1 ug) Data represents mean + SEM, n = 8

TABLE 3 Anti-angiogenesis efficacy of polycationic compound in CAM modelBranch pts ± % inhibition ± CAM Treatment SEM SEM PBS (control)  80 ± 7FgF2 (1.0 ug/ml)  177 ± 10 Compound 26 123 ± 7 55 ± 8 (1.0 ug) + FGF2(1ug) Compound 34 156 ± 9 21 ± 9 (1.0 ug) + FGF2(1 ug) Compound 40 142 ± 636 ± 6 (0.1 ug) + FGF2(1 ug) Compound 36 156 ± 6 21 ± 6 (1.0 ug) +FGF2(1 ug) Compound 33  160 ± 18  17 ± 18 (1.0 ug) + FGF2(1 ug)Ccompound 27 144 ± 9 34 ± 9 (0.1 ug) + FGF2(1 ug)

TABLE 4 Anti-angiogenesis efficacy of polycationic compound in CAM modelMean % Treatment Groups inhibition ± SEM PBS (Control FGF2 (1.0 ug/ml)Compound 50 (1.0 ug) + FGF2 (1 ug) 21 ± 9 Compound 50 (3.0 ug) + FGF2 (1ug) 42 ± 8 Compound 50 (10 ug) + FGF2 (1 ug) 66 ± 7 Compound 48 (1.0ug) + FGF2 (1 ug) 16 ± 6 Compound 48 (3.0 ug) + FGF2 (1 ug) 38 ± 8Compound 48 (10 ug) + FGF2 (1 ug) 55 ± 7 Data represent mean ± SEM, n =8

As depicted in Tables 2, 3 and 4 above, the polycationic compoundsblocked FGF2-induced angiogenesis in the CAM model of angiogenesis.

Example 2

The following example illustrates inhibition of endothelial cell tubeformation by polycationic compounds of the present invention.Differentiation by endothelial cells was examined using a methoddeveloped by Grant et al. (Grant et al., In Vitro Cell Dev. Biol.,27A:327-336 (1991). Matrigel® matrix, phenol-red free (commerciallyavailable from Becton Dickinson, Bedford, Mass.) was thawed overnight at4° C. Using cold pipette tips, 3.0 mg/well of Matrigel® matrix wasplaced in a cold twenty-four-multiwell plate. Matrigel® matrix wasallowed to polymerize during incubation at 37° C. for 30 minutes.

Human umbilical vein endothelial cells (HUVEC) were maintained at 37° C.with 5% CO₂ and 95% humidity in endothelial cell growth medium with 2%fetal bovine serum (EGM). The tube assay was performed in endothelialcell basal medium (EBM) supplemented with 0.5% bovine serum albumin(BSA) and 1:100 diluted Insulin-Transferrin-Selenium-G supplement(I-T-Se, 100×). HUVEC were trypsinized and centrifuged and,subsequently, washed twice in phosphate buffered saline (PBS). Aftercounting, cell density was adjusted to 35,000 cells/mL.

A final concentration of 35,000 cells/mL/well was treated withrecombinant human fibroblast growth factor basic (FGF2) at 100 ng/ml andpolycationic compounds (see Table 1B) dissolved in EBM medium. Treatedcells were incubated overnight at 37° C. with 5% CO₂ and 95% humidity toallow cell attachment.

Subsequently, the medium was aspirated and cells were fixed and stainedusing a modified HEMA-3 stain kit. Digital images of micro-titer wellsections were collected using a DKC5000 3-CCD color video camera system(Toshiba America, New York, N.Y.) and analyzed using Image-Pro Plussoftware (Media Cybernetics, Silver Spring, Md.). The area and majoraxis length of stained cells having a tubular morphology was measured onthe Matrigel® matrix surface (Becton Dickinson, Bedford, Pa.) countedfrom 5 images/well.

As illustrated in Table 5 below, polycationic compounds are potentinhibitors of EC tube formation in vitro.

TABLE 5 Anti-angiogenesis efficacy of polycationic compound in the humanendothelial tube formation assay Mean % Treatment Groups Inhibition ±SEM Compound 29 (0.01 ug) 25 ± 7 Compound 29 (0.1 ug) 42 ± 5 Compound 29(1.0 ug) 76 ± 6 Data represent mean ± SEM, n = 3

Example 3

This example relates to cellular migration assays. These assays wereperformed using a Neuroprobe 96 well disposable chemotaxis chamber withan 8 μm pore size. This chamber allowed for quantitation of cellularmigration towards a gradient of either vitronectin or osteopontin.Cultured cells were removed following a standardized method usingEDTA/Trypsin (0.01%/0.025%). Following removal, the cells were washedtwice and resuspended (2×10⁶/ml) in EBM (Endothelial cell basal media,Clonetics Inc.). Add either vitronectin or osteopontin (33 μl) at0.0125-100 μg/ml to the lower wells of a disposable chemotaxis chamber,and then assemble using the preframed filter. The cell suspension (45μl) was added to a polypropylene plate containing 5 μl of test agent atdifferent concentrations and incubated for 10 minutes at 22° C. Add 25μl of cell/test agent suspension to the upper filter wells then incubateovernight (22 hours at 37° C.) in a humidified cell culture incubator.After the overnight incubation, non-migrated cells and excess media weregently removed using a 12 channel pipette and a cell scraper. Thefilters were then washed twice in PBS (no Ca⁺² or Mg⁺²) and fixed with1% formaldehyde. Membranes of migrated cells were permeated with TritonX-100 (0.2%) then washed 2-3 times with PBS. The actin filaments ofmigrated cells were stained with rhodamine phalloidin (12.8 IU/ml for 30minutes (22° C.). Rhodamine phalloidin was made fresh weekly and reusedfor up to 3 days, when stored protected from light at 4° C. Chemotaxiswas quantitatively determined by fluorescence detection using aCytofluor II (530 excitation/590 emission). All cell treatment andsubsequent washings were carried out using a uniquely designedtreatment/wash station. This station consisted of six individual reagentunits each with a 30 ml volume capacity. Individual units were filledwith one of the following reagents: PBS, formaldehyde, Triton X-100, orrhodamine-phalloidin. Using this technique, filters were gently dippedinto the appropriate solution, thus minimizing migrated cell loss. Thistechnique allowed for maximum quantitation of cell migration andprovided reproducible results with minimal inter and intra assayvariability (Bozarth et al, Methods In Cell Science, 19 (3): 179-187,1997; Penno et al, J. Method In Cell Science, 19 (3): 189-195, 1997).

As illustrated in Table 6 below, the polycationic compounds of thepresent invention inhibit human umbilical vein endothelial migration.

TABLE 6 Effect of polycationic compound on human endothelial cellmigration assay Mean % Treatment Groups Inhibition ± SEM Compound 29(0.01 uM) 19 ± 2 Compound 29 (0.1 uM) 40 ± 3 Compound 29 (1.0 uM) 67 ± 5Data represent mean ± SEM, n = 3

Example 4

The following example illustrates the heparin antagonistic effects ofpolycationic compounds of the present invention. To determine theanti-heparin activity of the polycationic compounds an assay measuringthe percent inhibition using a fixed concentration of polycationiccompound or concentrations of polycationic compounds causing lysis of50% of human red blood cells were conducted.

10 IU of anti-thrombin was dissolved in 10 ml of buffer, resulting in a1 IU/ml stock solution (250×) of the anti-thrombin. The 1 IU/ml (250×)stock solution of anti-thrombin and a 336 mM stock solution of NaCl werediluted into a total volume of 50 μl buffer so that the finalanti-thrombin concentration was 0.004 IU/sample well and the NaCl was150 mM/sample well. 1 μl of the compound to be tested, finalconcentration 10 μg/ml (corresponding to 0.5 logarithmic antagonistdilution) is added to the sample well. The samples are mixed and allowedto incubate at room temperature for 20 minutes. 50 μl of factor Xadissolved in buffer is added to the sample well to a final concentrationof 0.14 knat/well (2 IA of the 7.1 knat/ml stock solution to a finalsample well buffer volume of 100 μl). The samples were mixed and furtherincubated at room temperature for 10 minutes. 10 μl of a 4 mM stocksolution of the substrate S-2765 was added to each sample well for afinal concentration of 0.4 mM in each sample well. The samples weremixed and hydrolyses of the chromogenic substrate Z-D-Arg-Gly-Arg-pNA(S-2765), thus liberating the chromophoric group pNA (p-nitroaniline),was monitored at 405 nm. The samples were mixed every 30 seconds tomaintain a uniform mixture. ThermoLabsystems Multiskan Spectrumspectrophotometer was used to measure the absorbance spectrums. Theincrease in absorbance was proportional to the enzyme (factor Xa)activity. The % inhibition of factor Xa was determined using a standardcurve. Results are depicted in Table 7. A bar graph also illustratingthe percent inhibition is presented in FIG. 2.

TABLE 7 % FactorXa Inhibition: Single concentration (10 ug/ml) Compound# % Inhibition Compound # % Inhibition 9 16.24689847 25 0 10 20.8014683426 48.14324 11 1.903402332 27 8.885942 12 9.381054349 28 44.29708 1336.84443085 30 49.96431121 24 1.835423677 31 75.45630672 5 39.767513 3223.1127426 19 59.82121614 33 32.01794636 15 5.710206995 34 99.9966010714 40.99112879 37 62.40440502 17 15.02328269 35 79.60300466 1813.25583767 38 65.05557255 1 22.29699874 39 56.49026206 2 41.05910744 417.817545291 4 0.951701166 46 59.14142959 3 2.855103498 42 79.46704735 62.583188879 43 59.68525883 7 5.506271031 45 77.83555963 8 7.409673363 4474.36864824 9 10.87658475 52 45.47772 20 7.851534618 53 43.03048843 211.495530403 54 19.98572448 22 1.291594439 55 46.49739982 23 1.223615785Magainin 4.418612556 16 30.38645865 Magainin-T 23.1127426

As illustrated in FIG. 2 and Table 7 above, the polycationic compoundsof the present invention inhibited Factor Xa.

Factor Xa Inhibition: EC50. To determine the concentration ofpolycationic compound that causes about 50% lysis of human red bloodcells, fixed heparin concentrations were used and different amounts ofheparin antagonists were added. Results are depicted in Table 8.

TABLE 8 FactorXa Inhibition: EC50 EC₅₀ HC₅₀ HC₅₀ Compound MW (μM)(μg/ml) μM) Compound 27 783 9.7 >2,000 Compound 25 615 5.3 >2,000Compound 26 927 2.0 >2,000 Compound 7 921 3.7 715 519 Compound 50 11260.36 637 377 Compound 48 1238 0.077 261 144 Compound 47 933 5.54Compound 51 1070 16.7 Compound 49 849 22

As illustrated in Table 7 above, the polycationic compounds of thepresent invention exhibit varying degrees of inhibition of Factor Xa.

Example 5

The following example illustrates the effect of a polycationic compoundof the present invention on coagulation time. The anti-heparin assay asdescribed herein was used. The assay contained either 1 mg/L, 2 mg/L or4 mg/l of heparin and increasing amounts of Compound 26 was added. Table9 and FIG. 3 depict the effect of Compound 26 on coagulation time.

TABLE 9 Effect of Compound 26 on coagulation time and heparin efficacyHeparin Compound 26 Coagulation Heparin (mg/L) (mg/L) Time (s) Efficacy1 0 50.8 1 1 1.25 42.8 0.65065 1 2 33.4 0.24017 1 2.5 31.3 0.14847 1 427.9 −1.67E−08 2 0 110.8 1 2 2.5 40.2 0.11083 2 4 33.9 0.031486 2 5 31.90.0062972 2 6 31.8 0.0050378 2 10 34.4 0.037783 4 0 214.9 1 4 2.5 124.80.51297 4 4 87.4 0.31081 4 5 55.8 0.14 4 6 35.4 0.02973 10 29.9−2.06E−09

As illustrated in FIG. 3 and Table 9, above, Compound 26, a polycationiccompound of the present invention decreased the coagulation time invarying concentrations of heparin, evidencing the compounds ability toantagonize heparin's activity.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow. Other embodiments of the invention will beapparent to those skilled in the art from a consideration of thisspecification or practice of the invention disclosed herein. It isintended that the specification and examples be considered as exemplaryonly, with the true scope and spirit of the invention being indicated bythe following claims.

1-24. (canceled)
 25. A compound of the formula:


26. A composition comprising the compound of claim 25 and a pharmaceutically acceptable carrier.
 27. The composition of claim 26 which is in the form of a capsule, cachet, tablet or lozenge.
 28. The composition of claim 26 which is in the form of a powder or granule.
 29. The composition of claim 26 which is in the form of a suspension or solution in an aqueous liquid or non-aqueous liquid.
 30. A method of antagonizing heparin in a mammal comprising administering the compound of claim 25 to said mammal.
 31. A method of antagonizing heparin in a mammal comprising administering the composition of claim 26 to said mammal.
 32. A method of inhibiting anti-Factor Xa in a mammal comprising administering the compound of claim 25 to said mammal.
 33. A method of inhibiting anti-Factor Xa in a mammal comprising administering the composition of claim 26 to said mammal.
 34. The method of claim 30 wherein the mammal is a human.
 35. The method of claim 31 wherein the mammal is a human.
 36. The method of claim 32 wherein the mammal is a human.
 37. The method of claim 33 wherein the mammal is a human. 